PROJECT SUMMARY/ABSTRACT The rod outer segment (OS) is a modified cilium containing ~1,000 stacked disc membranes that are densely packed with the visual pigment rhodopsin. The mammalian OS is renewed every 10 days; new discs are assembled at the bases of the OS by a poorly understood mechanism. The inner segment, where the biosynthetic and the endocytic pathways are housed, is linked with the OS via the axonemal connecting cilium. The precise route taken by rhodopsin from its site of synthesis to the disc membrane and the mechanism underlying disc renewal are not completely elucidated. However, these questions are not only biologically interesting, but are also clinically relevant because a large number of retinal degenerative diseases are manifested by rhodopsin mislocalization and disc disorganization. Our previous studies suggested that in mammalian rods the new discs are assembled and grown via Smad anchor for receptor activation (SARA)-mediated fusions between axonemal rhodopsin vesicles and nascent discs. The new concept that rhodopsin vesicles provide building blocks for disc membranes raises many questions. What is the nature of the vesicles? What are the molecular and regulatory pathways that underlie the production and the delivery of these vesicles? Because SARA is an early/sorting endosomal protein that directly binds to rhodopsin and the biosynthetic pathways of most apical surface proteins interact with the endocytic pathway, we will test the hypothesis that post- Golgi rhodopsins traverse the endocytic compartments en route to the OS (Specific Aim1). How rhodopsin's targeting is affected by strategies interfering with either the endocytic trafficking process or the functions of specific endosomal compartments will be examined. Furthermore, We will test the hypothesis that SARA also has a role in regulating the vesicular trafficking of rhodopsin in addition to its role in the disc fusion (Specific Aim 2). A number of mouse models in which sara gene can be deleted in rods under tissue-specific and/or temporally-regulated fashion will be employed in these studies. Finally, we will test our hypothesis that environmental lighting plays an important role in regulating the OS delivery, and, hence, disc incorporation of rhodopsin (Specific Aim3). Transfected rodent rods, conditional knockout mice, cell culture models, and several innovative techniques will be employed to comprehensively investigate these questions.